Bacteria are billions of years old and have developed a mechanism by which their DNA retains pieces of the virus so that if the virus reappears later, the infection stops by recognizing the pathogen and eliminating it from your body. This immune system of many bacteria and archaea is called CRISPR or genetic ‘cut and paste’, it allows the creation of genetic modifications in any organism and its usefulness has begun to be tested to treat different diseases, from breast cancer, autism or epidermolysis bullosa (butterfly skin).
CRISPR uses guides and a protein (Cas9 nuclease) to target certain regions of DNA and cut. After this, and naturally, the ends are glued together and the gene is inactivated. The problem facing scientists is that we are very familiar with most of the bacteria for which this mechanism is known because they are in our immediate environment and we are immunized to their system, so those proteins do not work with us.
For this reason, scientists have been looking for a long time in remote areas, such as Antarctica, the top of Everest or the Mariana Trench, trying to find bacterial species with which we have not had contact and whose proteins (Cas nucleases) work in U.S. However, a team of Spanish researchers has taken a different approach to this search: “Instead of looking in space, we have searched in time,” he explained on RTVE. Lluís Montoliu, researcher and deputy director of the National Center for Biotechnology (CNB-CSIC) and the Center for Network Biomedical Research in Rare Diseases (CIBERER-ISCIII) and one of those responsible for the study. “We’ve kind of ‘back to the future’ looking for different nucleases.”
How the immune system works in bacteria from millions of years ago
The biologist Francis Mojica gave CRISPR its name as part of his studies of microbes that lived in the hostile environment of the salt flats of Santa Pola (Alicante) in the early 1990s, and also analyzed other key sequences called PAM, which they allow the microbe to distinguish between the genome of a virus and its own since, without PAMs, a bacterium could eliminate itself. The study shows that the oldest Cas cut without the need for PAM. Mojica, co-author of the current work, highlights its importance for understanding the origin and evolution of CRISPR.
“This scientific achievement opens new avenues in the manipulation of DNA and treatment of diseases such as ALS, cancer, diabetes, or even as a diagnostic tool for diseases”
“Thanks to this reconstruction, we see how the immune system of microbes became less harmful to their carriers and increasingly specific for each virus,” he explains. Furthermore, “this work is important because it opens up a huge toolbox for creating better CRISPR systems,” he says.
With the aim of discovering how this ancient bacterial immune system was formed, a team made up of some of the leading experts in gene editing in Spain used a technique known as ancestral sequence reconstruction, which reconstructs the genome of extinct organisms using powerful computers to compare the complete genomes of current living beings – which contain billions of DNA letters – and estimate what the genome of their common ancestors would be like.
And this is how researchers have been able to travel back in time to recover Cas proteins found in extinct microbes; the oldest are 2.6 billion years old. In addition, they have also rescued extinct proteins from microorganisms that lived 1,000 million, 200 million, 137 million and 37 million years ago. Using these proteins, they have created new CRISPR systems and injected them into human cells. The results –which have been published in Nature Microbiology– show that all these proteins, despite their age, are capable of editing the genome.
Medical applications of these ancient proteins
In the laboratory, researchers have been able to observe something similar to fast-forward evolution. The oldest protein of all can only cut single-stranded DNA, whereas human DNA is made up of double-stranded DNA, but the other newer Cas molecules can cut human DNA effectively, and indeed have been able to. correct two genes, TYR and OCA2, that cause albinism.
Raúl Pérez-Jiménez, a researcher at the NanoGUNE Basque cooperative research center in nanoscience and co-author of the study, highlighted the potential of this research: “These are the oldest Cas proteins that have ever been obtained. We think they are like a diamond in the rough. Now we are going to study how we can make them as efficient as the current ones or even better”, he indicates.
Ylenia Jabalera, project researcher at nanoGUNE, maintains that “this scientific achievement makes it possible to have genetic editing tools with properties different from the current ones, much more flexible, which opens up new avenues in the manipulation of DNA and treatment of diseases such as ALS , cancer, diabetes, or even as a disease diagnostic tool”.
Miguel Ángel Moreno Pelayo, head of genetics at the Ramón y Cajal Hospital in Madrid and co-author of the work, highlights that the reconstruction of ancient proteins opens the possibility of designing new forms of synthetic CRISPR “that do not exist in nature.” Among other projects, his team develops this type of molecule to try to correct genetic defects in patients with amyotrophic lateral sclerosis. “We are facing a new paradigm”, sums up the scientist.
Lluís Montoliu highlights another advantage of the primitive Cas proteins. The potential for gene editing of the CRISPR system was discovered in bacteria of the species S. pyogenes. These microbes can cause infections, so many people have antibodies that can trigger immune reactions against the CRISPR extracted from them. The primitive Cas, on the other hand, are very different from any current version, so they are not detected by the immune system, a great advantage to avoid rejection in future medical applications, explains Montoliu.
This scientist explains that the reason why eukaryotes, the large group of multicellular organisms to which humans belong, did not develop a CRISPR-based immune system is because it was dangerous. “The most primitive CRISPR systems already allowed DNA to be cut, but they were very unselective, which probably ended up killing the organism they were trying to protect. In the world of bacteria, the individual is not important, what matters is the population, and this system allowed them to evolve and perfect an immune system even at the price of killing many along the way”, he concludes.
The study findings open up avenues for numerous medical applications. Miguel Ángel Moreno-Mateos, professor at the Pablo de Olavide University and Ramón y Cajal researcher at the Andalusian Center for Developmental Biology (CABD) opines: “It is a genuinely original work with an imaginative approach to, ultimately, understanding evolution of the CRISPR-Cas9 system for billions of years. The improvement of CRISPR-Cas technology requires a good understanding of how these systems work from a biochemical and structural point of view. Knowing how they have changed throughout evolution will allow us to approach these improvements from a new point of view, with potential applications in biotechnology and biomedicine. The study lays a foundation for now on them to continue working and being able to use this knowledge in tangible biotechnological applications. In any case, these basic studies are essential for progress in more applied sciences”, according to what he declared to SMC Spain.
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